The cardiac sarcolemmal Na-Ca exchanger (NCX) is the primary mechanism for the extrusion of Ca from myocytes. As such, the exchanger is an important regulator of intracellular Ca and cardiac contractility. The continuing long-term objective of this renewal is to further understand the significance of NCX in excitation contraction (EC) coupling and pacemaker activity. Genetic alterations in the level of NCX have had unexpected effects that have informed the field about novel aspects of EC coupling in cardiac cells. The three proposed projects are as follows: 1. Sinoatrial Node Activity in Atrial-Specific NCX Knockout Mice. We have recently produced novel atrial-specific NCX knockout (KO) mice that live into adulthood, but exhibit profound bradycardia and sinaotrial node (SAN) dysfunction. In both intact ex vivo SAN preparations and in isolated SAN cells, we will study the underlying mechanism for these adaptations, including changes in cellular electrophysiology and subcellular Ca movements. These experiments will resolve fundamental disputes in cardiac pacemaker generation theory and may have an enormous impact on the field. 2. Adaptations of Atrial Excitation-Contraction Coupling to Genetically Altered Levels of NCX. We will test the hypothesis that critical adaptations in atrial Ca regulation and EC coupling occur when atrial NCX is knocked out. Since atrial myocytes lack the spatial constraints associated with transverse-tubules of ventricular cells, we can test the hypothesis that regulation of EC coupling by NCX depends on spatial gradients and cellular microdomains. These studies will provide us with important insights into regulation of contractility in the atrium that may have important therapeutic implications. 3. Adaptations of Ventricular Excitation-Contraction Coupling to Genetically Altered Levels of NCX. We have produced ventricular-specific NCX KO mice that survive into adulthood. Ventricular myocytes from these mice tolerate ablation of NCX by reducing Ca entry through L-type Ca channels, yet maintain contractility by increasing EC coupling gain without increasing sarcoplasmic reticulum Ca load. We have proposed that gain increases because lack of NCX elevates diadic cleft Ca, and that elevated cleft Ca increases EC coupling fidelity. This suggests that cleft Ca manipulation by NCX could be used as an alternative to -agonists as an inotropic strategy that avoids cellular Ca overload and its deleterious consequences. The experiments in this aim will fill critical gaps in our understanding of the mechanism through which NCX ablation raises EC coupling gain without provoking Ca overload and cell death. This knowledge is essential so that we can learn how to manipulate NCX and cleft Ca as therapeutic targets in patients with heart failure and ischemic cardiac dysfunction.